Suspending medium for immunologic reactions

ABSTRACT

A low ionic strength suspending medium for immunologic reactions which includes a salt solution, a buffer, gelatin, albumin, and an organic solute to control osmolality.

BACKGROUND OF THE INVENTION

The present invention relates to a suspending medium for use inimmunologic reactions. More particularly, the invention relates to asuspending medium for immunohematologic reactions and to a method forpotentiating agglutination reactions.

Immunohematologic agglutination reactions have conventionally beenconducted in media comprised of physiological saline (ca.O.15M Na Cl) oralbumin solutions. Thus, in antibody screening, antibody identification,crossmatches, direct or indirect antiglobulin testing, blood grouping,and the like, cells are conventionally washed and suspended inphysiological saline for reaction with test serums. The use ofphysiological saline in such reactions suffers from certaindisadvantages. Notably, lengthy incubation times are often required toobserve agglutination reactions. In certain situations, such ascrossmatching for emergency transfusions, it is important to obtainreaction results as quickly as possible. Another disadvantage ofphysiological saline is that cells tend to be sticky and, thus, adhereto glass surfaces, making evaluation of reaction results difficult.

Physiological saline has been modified to overcome some of thesedisadvantages. Esposito, V.M. and Weinstein, S.B., U.S. Pat. No.3,970,427, describe a suspending solution containing physiologicalsaline and a small amount of gelatin. The addition of gelatin was foundto reduce or eliminate the problem of sticking between erythrocytes andglass surfaces, and to reduce hemolysis. Another procedure in widespreaduse involves the addition of an albumin solution, e.g. 22% or 30% bovinealbumin, to a suspension of cells in physiological saline. The albuminis thought to enhance the agglutination reaction by reducing theelectrostatic charge on the cell surfaces. Technical Manual of theAmerican Association of Blood Banks, Wm. V. Miller, Ed., AmericanAssociation of Blood Banks, Washington, D.C. 1977, p. 167.

For many years, low ionic strength solutions, i.e. solutions havingionic strengths lower than that of physiological saline, have been knownto enhance agglutination in many immunologic tests. However, theprocedures employing such solutions produced an unacceptable incidenceof false (non-antibody) positive reactions and stymied their generalacceptance. Low, B. and Messeter, L., Vox. Sang. 26, 53-61 (l974)reported a low ionic strength solution which included sodium glycinate.That solution produced few false positive reactions, and when comparedto standard procedures employing physiological saline or bovine albumin,a reduction in required incubation times with specificantibody-containing serums was realized. The solution also increased theantibody uptake and the strengths of agglutination reactions withcertain antibodies. Subsequent work indicates that procedures employinglow ionic strength suspending media are at least as sensitive, and inmost cases are more sensitive, than routine procedures employingphysiological saline or albumin for the detection of IgG and IgMantibodies.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an improved low ionicstrength solution for use in immunologic reactions. A further object ofthe invention is to provide a low ionic strength suspending medium whichenhances immunohematologic agglutination reactions.

In accordance with the invention, disclosed is an aqueous suspendingmedium for immunologic reactions, comprising gelatin, at a concentrationof from about 0.5 wt. % to about 1.5 wt. %; albumin, at a concentrationof from about 4.0 wt. % to about 6.0 wt. %; sufficient organic solute toprovide an osmolality of from about 150 mOsm/kg. H₂ O to about 450mOsm/kg. H₂ O; sufficient sodium chloride, potassium chloride, or amixture thereof to provide an ionic strength equivalent to about a 0.01molar to about a 0.10 molar solution of sodium chloride; and a pH fromabout 6.0 to about 8.0.

DETAILED DESCRIPTION OF THE INVENTION

The suspending medium of the present invention has a low ionic strength,as compared to physiological saline. Sodium or potassium chloride or amixture thereof is usually added to the solution to provide the desiredionic strength, but ions from other sources, such as buffers, pHadjusting agents, and albumin, also contribute to the ionic strength ofthe final product. Therefore, to determine the quantity of sodium orpotassium chloride to be added, the electrical conductivity (orresistivity ) of the solution may be used as a measure of ionicstrength. The absolute value of the electrical conductivity is notcritical, but the measurement is useful for comparison to solutions ofknown ionic strengths. The ionic strength of the suspending medium ofthe present invention is equivalent to about a 0.01 molar to about 0.1molar aqueous sodium chloride solution. Preferably, the ionic strengthwill be equivalent to about a 0.02 molar to about 0.05 molar sodiumchloride solution. It is not necessary to measure the electricalconductivity of each batch of solution, once the desired formula isdetermined, but the measurement is useful for initially determining therelative proportions of ingredients for a specific formulation.

The solution includes gelatin at a concentration of from about 0.5 wt. %to about 1.5 wt. %, preferably from about 0.7 wt. % to about 1.3 wt. %.The gelatin used in this invention is advantageously finely divided tofacilitate dissolution, and is preferably of at least laboratory gradepurity in accordance with the listing in the U.S. Pharmacopea orNational Formulary.

The source of the gelatin and the procedures used in its preparation arenot critical. Thus, gelatin obtained from the partial hydrolysis ofcollagen derived from calfskin, pigskin, and the skin, white connectivetissue, and bones of various animals all appear suitable. Similarly, thegelatin may be derived from an acid-treated precursor (Type A) or froman alkali-treated precursor (Type B).

Albumin is also advantageously dissolved in the solution at aconcentration of from about 3.0 wt. % to about 7.0 wt. %, preferablyfrom about 4.0 wt. % to about 6.0 wt. %. The source of the albumin usedis not critical. Bovine serum albumin is preferable because of itsavailability and cost, but albumin obtained from the fractionation ofplasma from virtually any animal, such as horse, sheep, swine, chickenand human, may be employed. The albumin is preferably substantiallysalt-free or low in salt, so as not to contribute appreciably to theionic strength of the final solution. The effect of the saltconcentration of the albumin on ionic strength should be considered whendetermining the concentration of other ingredients.

Because the ionic strength of the solution of the present invention islower than that of physiological saline, the osmolality must be adjustedto approximate physiological values. If the osmolality of the solutionis too low, the erythrocytes will lyse, and if it is too high, they willbecome crenated. The osmolality of the present solutions is controlledwithin a range of from about 150 mOsm/kg. H₂ O to about 450 mOsm/kg. H₂O. Preferably, the osmolality is adjusted from about 250 mOsm/kg. H₂ Oto about 400 mOsm/kg. H₂ O. Any solute which does not substantiallyincrease the ionic strength of the solution, and which does notdeleteriously react with any other components of the solution orinterfere with the immunologic reaction may be employed to controlosmolality. Such solute is preferably organic, such as an amino acid, asugar, a soluble alcohol, etc. Particularly preferred solutes areglycine, sucrose, and glucose.

The optimum pH for immunologic reactions varies from one antibody orantibody group to another. For a universal suspending medium, the pH isadvantageously adjusted to provide near optimum conditions for thegreatest number of tests. Accordingly, the pH of the suspending media ofthis invention is adjusted from about 6.0 to about 8.0. A preferred pHrange is from about 6.4 to about 7.4. The pH may be adjusted with anacid or a base as is well known in the art. Mineral acids such ashydrochloric acid, sulfuric acid, and the like and alkali or alkalineearth metal hydroxides such as sodium hydroxide, potassium hydroxide,etc. are used. To provide greater control of pH, a suitable buffer maybe used in a pH-controlling amount. Any buffer capable of controlling pHwithin the desired range and which does not interfere with the reaction,may be employed. Conventional phosphate buffers are preferred for thispurpose.

The ingredients of the suspending medium may be combined in anyconvenient manner. A preferred manner of preparing the solution is tosequentially dissolve the ingredients in a portion of the total volumeof water to be used. The water may be heated, e.g. to 50° C.-60° C. andstirred to facilitate dissolution of the gelatin. After the gelatin isdissolved, the remaining ingredients are dissolved in the solution. ThepH is then adjusted and the remaining water is added.

The solution will usually be packaged and stored for extended periods oftime; therefore, it is desirable to sterilize the solution and toincorporate a preservative therein. Sterilization may be accomplished byany convenient means such as sterile filtration, irradiation, and thelike. Suitable preservatives include conventional bacteriostats orantibiotics such as preserving amounts of thimerosal, phenylmercuricacetate, sodium azide, neomycin or chloramphenicol, or combinationsthereof.

The solutions of this invention may be employed in a wide variety ofimmunologic reactions. They are particularly useful in immunohematologicreactions, such as antibody screening, antibody identification,crossmatches, direct or indirect antiglobulin testing, blood grouping,and the like. The solutions may also be used in other types ofimmunologic reactions, such as latex particle agglutination tests, e.g.,as in the latex agglutination assay for hepatitis surface antigen, andother antibody/antigen reactions.

The present invention constitutes a significant advance over previoussuspending media for hematologic reactions. The addition of gelatin to alow ionic strength solution was found to improve the performance of thatsolution. Agglutination reactions were stronger and more easilyinterpreted, and incubation times were reduced.

Surprisingly, the incorporation of both albumin and gelatin into theformulation resulted in a suspending medium having properties muchimproved over conventional albumin or saline solutions, orglycine-containing low ionic strength solutions. Sensitivity was greaterthan that of the solution containing only gelatin, resuspension wasfacilitated, and there were fewer questionable or rough looking negativereactions. Moreover, the incidence of nonspecific agglutinationreactions or false positives has been found to be quite low with thesesolutions.

In addition to its use in antibody detection, antibody identification,and compatibility testing, the suspending medium of the presentinvention may be utilized as the base for preparation of various bloodgrouping serums. These serums have the advantages of increasedsensitivity and decreased reaction times that are found using low ionicstrength solutions with patient antibodies. The increased sensitivityallows less antiserum (or lower titers) to be used for preparation ofblood grouping serums of at least equal reactivity to conventionalserums. The cost savings associated with such serums is apparent.

Thus, a new low ionic strength solution has been discovered whichpossesses antibody-enhancing characteristics superior to the solutionscurrently used in blood banking. Increased sensitivity is realized whilethe incubation times required for both IgG and IgM antibodies arereduced.

The invention is further illustrated by the following examples which arenot intended to be limiting.

EXAMPLE I

Approximately 400 kg. of purified water was charged into a stainlesssteel tank equipped with an agitator and a heating means. The water washeated to 50° C.-60° C. Gelatin (8.0 kg.) was added to the water and themixture was stirred for 2-21/2 hours at 50° C.-60° C. The temperaturewas maintained while albumin (343 kg. of 15% solution), sodium chloride(716 g), glycine (21.6 kg.), sodium phosphate dibasic (85 g), sodiumphosphate monobasic (83 g), and thimerosal (100 g) were dissolved. ThepH of the solution was adjusted to 6.9±0.1 with 1N sodium hydroxide, andwater was added to a final weight of 1019 kg. The solution was sterilefiltered while hot and filled into bottles.

EXAMPLE II

The suspending medium described in Example I was tested by blood banklaboratories in over 1700 assays involving identification ofrepresentative samples of all major blood groups. The tests weredesigned to confirm the efficacy of the solution and compare it tostandard procedures employed by blood banks. Cells and serums wereobtained from patient samples routinely submitted to the laboratory. Noselection of patient population by age, sex, medication, disease state,etc. was made. The basic testing performed on patient samples includedan autocontrol consisting of the patients' cells with autologous serum,a direct antiglobulin test on patients' cells and an antibody screen onthe patients' serum. If the antibody screen was positive, an antibodyidentification panel was employed to attempt to identify the specificityof the antibody.

Assays were conducted by adding to a test tube 2 drops of patient serum,1 drop of a 3%-5% saline cell suspension, and 4 drops of the suspendingmedium. Where sufficient patient sample was available, two tubes wereprepared for each sample. One tube was centrifuged immediately, examinedimmediately after centrifugation, after 10 minutes at room temperature,and again after 5 minutes at 5° C. The second tube was examined after 15minutes at 37° C. and again after washing and addition of anti-human(Coombs) serum. When insufficient sample was available for two tubetesting, one tube was used. The tube was examined immediately aftercentrifugation, after room temperature incubation (10 minutes), afterincubation at 37° C. for 15 minutes, and again after washing andaddition of the anti-human serum.

Parallel samples were assayed by standard methods conventionallyemployed in the laboratory. These involved either a saline method,employing physiological saline or a "high protein" method, employing 22%or 30% albumin. In the saline procedure each tube received 2 drops ofpatient serum and 1 drop of 3%-5% saline cell suspension. In the highprotein procedure, each tube received 2 drops of patient serum, 3 dropsof albumin solution, and 1 drop of 3%-5% saline cell supension.

Reactions were observed using some type of optical aid such as anagglutination viewer. The reactions were read macroscopically and gradedas follows:

4+=One large cell button that remains intact on gentle resuspension.

3+=Several large agglutinates of cells on gentle resuspension.

2+=Many small agglutinates of approximately equal size, clearbackground.

1+=Many small but definite agglutinates, finely granular appearance,opaque reddish background.

±=Minute agglutinates, many unagglutinated cells.

h=Hemolysis.

-=Negative reaction. No agglutination.

Interpretation:

Positive-Cells agglutinated

Negative-No agglutination.

Intermediate gradations using superscript "s" for stronger and "w" forweaker were used; e.g., 1^(s),1^(w), etc., creating a total of twelveagglutination strengths (12 point scale).

The results of the tests were reported and grouped according to eachantibody. The data was summarized and comparisons to standard tests weremade. The performance of the suspending medium of the present inventionas compared to standard procedures are listed in Table I. The firstcolumn of Table I indicates the particular antibody/antigen reactionobserved, column 2 indicates the point at which an observation was made:LT=after five minutes incubation at 5° C., RT=after 10 minutesincubation at room temperature, 37=after 15 minutes incubation at 37°C., AG=after cell washing and addition of anti-human serum. Columns 3and 4 indicate the performance of the solution of present invention tostandard procedures for antibody screen results and panel results. Inthe subcolumn under >Std., the percentage of expected positive reactionswhich were significantly stronger than the standard procedure (i.e.having a reaction strength separated by at least a 1+difference) arelisted. Likewise, the subcolumn under=Std. lists the percentage ofexpected positive reactions for the test medium equivalent to thestandard procedure (i.e. separated by less than 1+difference), and thesubcolumn under<Std., lists the percentage of expected positivereactions for the test medium significantly weaker than the standardprocedure (separated by greater than 1+difference). Note that a positivetest is always considered significant over negative test even if thereaction strengths differ by less than 1+. A positive reaction isdefined as any reaction having a strength of ± or greater.

Although the performance of the suspending medium of this inventionvaried from one antibody to another, overall, the solution performedsignificantly better than standard procedures. Each antibody/antigengroup was assayed at all four phases for evaluation purposes. Undernormal testing conditions, those skilled in the art recognize thatcertain phases are more reliable for antibodies having certain bloodgroup specificities. In Table I, the phases conventionally used forparticular antibodies are indicated by asterisks.

EXAMPLE III

In this example, a procedure employing the suspending medium describedin Example I was compared to procedures employing low ionic strengthsaline plus glycine, low ionic strength saline plus albumin, andstandard procedures as described in Example II.

Serums were selected based on prior data indicating the presence ofspecific antibodies, without regard to patient sex, age, disease status,etc. Red cells were commercial reagent screening cells having knownantigenic compositions. Reactions were rated on a twelve point system asdescribed in Example II except that reaction strengths were expressed ona linear scale from negative (0) to 12(4+).

The experimental results are reported in Table II. Solution A is thesuspending medium described in Example I. Solution B is a commercialphosphate buffered low ionic strength saline solution containing glycineand a preservative. Solution C is a commercial phosphate buffered lowionic strength saline solution containing 5% albumin. The StandardMethod involves initial reaction in physiological saline, followed byaddition of 3 drops of 30% albumin for the 37° C.incubation, thenconversion to antiglobulin (Coombs) testing.

All assays were one tube tests. Procedures using the suspending mediumdescribed in Example I were conducted as described in Example II. Inprocedures using Solution B, cells were washed with physiological salineand a 3% cell suspension in Solution B was prepared. Two drops of serumwas added to one drop of the cell suspension. The cells were centrifugedand observed after 10 minutes incubation at room temperature,resuspended and reexamined after incubation at 37° C. for 15 minutes,and then converted to antiglobulin (Coombs) testing. In procedures usingSolution C, two drops of serum were mixed with one drop of a 3-5%suspension of cells in physiological saline and four drops of Solution Cwere added. The reactions were examined in the same manner as withSolution B.

The reaction scores listed in Table II indicate that the suspendingmedium of the present invention results in greater sensitivity than theother media, and the test totals demonstrate an overall superiority.

                  TABLE I                                                         ______________________________________                                        Anti-    Antibody Screen Results                                                                       Panel Results                                        body Phase   >Std    =Std  <Std  >Std  =Std  <Std                             ______________________________________                                        D    LT                                                                            RT      7       7     7     0     0     8                                     37      0       0     29    5     0     26                                    AG*     50      43    0     43    52    4                                C    LT                                                                            RT                                                                            37                                                                            AG*                         60    0     0                                --c  LT                                                                            RT                          3     22    0                                     37                          3     22    0                                     AG*     50      50    0     19    63    3                                E    LT      33      0     0     15    7     0                                     RT      38      0     0     69    6     3                                     37      13      25    13    13    26    3                                     AG*     25      75    0     54    77    0                                --e  LT                                                                            RT                                                                            37                          0     0     45                                    AG*                         0     90    10                               M    LT      0       0     0     3     39    0                                     RT*     75      25    0     66    10    3                                     37      25      0     0     50    6     1                                     AG      0       25    25    22    37    3                                N    LT                          30    0     0                                     RT*                         33    48    15                                    AG                                                                       S    LT                                80    20                                    RT*                               100                                         37                                100                                         AG                                100                                    P.sub.1                                                                            LT      0       25    0     20    74    6                                     RT*     25      75    0     22    52    7                                     37      25      0     75    0     26    7                                     AG*     0       25    50    9     37    11                               Le.sup.a                                                                           LT                                                                            RT*     8       42    25    6     39    19                                    37      8       17    50    7     65    31                                    AG*     13      33    50    9     40    24                               Le.sup.b                                                                           LT                                                                       RT   17      22      6     16    41    22                                          37      11      11    22    12    27    25                                    AG*     6       17    17    13    35    21                               K    LT      0       0     17    30    10    0                                     RT      8       33    8     6     36    3                                     37      8       33    0     6     25    0                                     AG*     33      67    0     17    78    3                                --k  LT                                                                            RT                                                                            37                                                                            AG*                         80    20    0                                K.sub.p.sup.a                                                                      LT                                                                            RT                                                                            37                                                                            AG*                         0     67    33                               F.sub.y.sup.a                                                                      LT                                                                            RT                                                                            37                                                                            AG*                         35    65    0                                ______________________________________                                    

                                      TABLE II                                    __________________________________________________________________________              10 minutes        15 minutes        Antiglobulin                    TEST PHASE→                                                                      Room Temperature  37° C.     Serum                                     Sol'n.                                                                            Sol'n.                                                                            Sol'n.                                                                            Standard                                                                            Sol'n.                                                                            Sol'n.                                                                            Sol'n.                                                                            Standard                                                                            Sol'n.                                                                            Sol'n.                                                                            Sol'n.                                                                            Standard            TEST METHOD→                                                                     A   B   C   Method                                                                              A   B   C   Method                                                                              A   B   C   Method              __________________________________________________________________________     Antibody                                                                     Anti-D    10  3   6   0     9   3   6   6     10  11  10  8                   Anti-D    0   0   0   0     0   0   0   0     9   6   9   3                   Anti-D + C                                                                              0   0   0   0     3   0   0   0     11  10  11  9                   Anti-E    0   0   0   0     3   0   1   1     0   1   0   0                   Anti-e    3   0   4   0     0   0   0   3     3   0   3   3                   Anti-M    10  5   12  0     9   1   9   1     8   0   4   3                   Anti-S    6   0   3   0     3   0   0   0     3   0   3   2                   Anti-P.sub.1                                                                            6   3   5   3     5   3   0   0     3   1   1   3                   Anti-Le.sup.a                                                                           10  9   9   7     6   3   4   3     1   3   0   3                   Anti-Le.sup.bH                                                                          6   4   0   0     0   0   0   0     0   0   0   0                   Anti-K    3   0   0   0     3   0   0   0     11  9   11  12                  Anti-Fy.sup.a                                                                           3   4   3   0     4   3   7   0     5   5   3   6                   Anti-Jk.sup.a                                                                           0   0   0   0     0   0   0   0     9   11  9   3                   Total Score                                                                             47  28  42  10    45  10  27  14    73  57  64  55                  __________________________________________________________________________

We claim:
 1. A suspending medium for an immunologic reaction, comprisinggelatin, at a concentration of from about 0.5 wt. % to about 1.5 wt. %;albumin, at a concentration of from about 4.0 wt. % to about 6.0 wt. %;sufficient organic solute to provide an osmolality of from about 150mOsm/kg. H₂ O to about 450 mOsm/kg. H₂ O; sufficient sodium chloride,potassium chloride, or a mixture thereof to provide an ionic strengthequivalent to about a 0.01 molar to about a 0.10 molar solution ofsodium chloride; and a pH from about 6.0 to about 8.0.
 2. The suspendingmedium of claim 1 wherein the gelatin concentration is from about 0.7wt. % to about 1.3 wt. %.
 3. The suspending medium of claim 1 whereinthe gelatin concentration is from about 0.7 wt. % to about 1.3 wt. % andthe albumin concentration is from about 4.0 wt.% to about 6.0 wt.%. 4.The suspending medium of claim 3 wherein the albumin is substantiallysalt-free bovine serum albumin.
 5. The suspending medium of claim 1wherein the osmolality is from about 250 mOsm/kg. H₂ O to about 400mOsm/kg. H₂ O.
 6. The suspending medium of claim 3 wherein theosmolality is from about 250 mOsm/kg. H₂ O to about 400 mOsm/kg. H₂ O.7. The suspending medium of claim 6 wherein the organic solute is anamino acid, a sugar, or a water soluble alcohol.
 8. The suspendingmedium of claim 6 wherein the organic solute is glycine, sucrose, orglucose.
 9. The suspending medium of claim 6 wherein the organic soluteis glycine.
 10. The suspending medium of claim 1 wherein the pH is fromabout 6.4 to about 7.4 .
 11. The suspending medium of claim 3 whereinthe pH is from about 6.4 to about 7.4.
 12. The suspending medium ofclaim 8 wherein the pH is from about 6.4 to about 7.4.
 13. Thesuspending medium of claim 1, further comprising a pH-controlling amountof a buffer.
 14. The suspending medium of claim 13 wherein the buffer isa phosphate buffer.
 15. The suspending medium of claim 3, furthercomprising a pH-controlling amount of a phosphate buffer.
 16. Thesuspending medium of claim 8, further comprising a pH-controlling amountof a phosphate buffer.
 17. The suspending medium of claim 1, furthercomprising a preserving amount of a bacteriostat or an antibiotic. 18.The suspending medium of claim 17, wherein the antibiotic is neomycin orchloramphenicol, or the bacteriostat is thimerosal, phenylmercuricacetate, or sodium azide.
 19. The suspending medium of claim 3, furthercomprising a preserving amount of thimerosal.
 20. The suspending mediumof claim 8, further comprising a preserving amount of thimerosal. 21.The suspending medium of claim 16, further comprising a preservingamount of thimerosal.
 22. The suspending medium of claim 1, 3, 4, 8, 9,12, 16, or 20, wherein the ionic strength is equivalent to about a 0.02molar to about 0.05 molar sodium chloride solution.
 23. The suspendingmedium of claim 22 wherein the immunologic reaction is animmunohematologic reaction.